Structure and Function of CTCF: Mouse Model Studies

CTCF的结构和功能：小鼠模型研究

• 批准号: 6680238
• 负责人: GALINA N FILIPPOVA
• 金额: $ 39.97万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-05 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6680238
• 关键词: chromosomes; embryogenesis; flow cytometry; gene expression; gene targeting; genetic promoter element; genetic regulation; genetically modified animals; immunocytochemistry; laboratory mouse; methylation; neoplasm /cancer genetics; northern blottings; polymerase chain reaction; protein structure function; southern blotting; tissue /cell culture; transcription factor; tumor suppressor proteins

DESCRIPTION (provided by applicant): CTCF is a highly conserved 11 Zn finger (ZF) transcription factor and insulator protein. CTCF is involved in multiple aspects of gene regulation including promoter activation and repression, hormone-responsive gene silencing, methylation-dependent chromatin insulation, and genomic imprinting. Moreover, our recent discovery of a methylationsensitive CTCF insulator at the DM1 locus that might be responsible for the severe phenotype of Congenital Myotonic Dystrophy (Filippova et al.,2001), and our preliminary data on the role of CTCF insulators in X chromosome inactivation strongly suggest a link between CTCF and epigenetic regulation in development. Several findings also point to a role for CTCF as a tumor suppressor gene in both cancer epigenetics and genetics. Aberrant methylation of certain CTCF target sites, that is predicted to prevent CTCF-binding, has been demonstrated in a number of tumors. CTCF maps to human chromosome 16q22, within a region that displays frequent cancer-associated deletions (Filippova et al.,1998). We have observed somatic missense mutations within the CTCF 11ZF DNA-binding domain in breast, prostate and Wilms' tumors (Filippova et al.,2002). CTCF +/- mice exhibit enhanced tumor development in multiple tissues. Homozygous deletion of the CTCF results in early embryonic lethality, whereas the CTCF heterozygous mice exhibit decreased embryonic survival. Our broad and longterm hypothesis is that certain functions of CTCF are essential in early development and maintaining cell viability, whereas its other functions, when lost, result in the malignant phenotype. To test this hypothesis our specific aims will: Extend our studies on the role of CTCF in tumorigenesis (Aim 1) and early embryonic development (Aim 2). We will determine what genetic and epigenetic mechanisms are involved in CTCF haploinsufficiency. And finally (Aim 3), we will use the Cre-loxP system to generate a conditional CTCF mutant allele, that in combination with various tissue-specific Cre transgenic mice will allow us to test the hypothesis that complete loss of CTCF function leads to cell death in both normal and/or malignant cells, whereas loss of one CTCF allele in somatic tissue predisposes to tumor development. We will also generate knock-in mice harboring CTCF Zn finger point mutations that we have observed in human tumors to determine the functional significance of such mutated alleles in tumor predisposition. The significance of this proposal is that identifying the mechanisms of the CTCF haploinsufficiency will provide new insight into the broad spectrum of clinical human cancer phenotypes associated with LOH at 16q22.1 where CTCF maps. The health-relatedness is that the identified mechanisms can be targeted for both cancer prevention and therapeutic intervention.

描述（由申请人提供）：CTCF是高度保守的11锌指（ZF）转录因子和绝缘子蛋白。CTCF参与基因调控的多个方面，包括启动子激活和抑制、转录响应性基因沉默、甲基化依赖性染色质绝缘和基因组印记。此外，我们最近在DM 1基因座发现了甲基化敏感性CTCF绝缘子，其可能是先天性肌强直性营养不良症的严重表型的原因（Filippova et al.，2001），我们关于CTCF绝缘子在X染色体失活中的作用的初步数据强烈表明CTCF和发育中的表观遗传调控之间的联系。一些研究结果还指出CTCF作为肿瘤抑制基因在癌症表观遗传学和遗传学中的作用。某些CTCF靶位点的异常甲基化（预计会阻止CTCF结合）已在许多肿瘤中得到证实。CTCF定位于人染色体16 q22，在显示频繁的癌症相关缺失的区域内（Filippova et al.，1998年）。我们已经在乳腺、前列腺和Wilms肿瘤中观察到CTCF 11 ZF DNA结合结构域内的体细胞错义突变（Filippova et al.，2002年）。CTCF +/-小鼠在多种组织中表现出增强的肿瘤发展。CTCF的纯合缺失导致早期胚胎死亡，而CTCF杂合小鼠表现出降低的胚胎存活率。我们广泛和长期的假设是，CTCF的某些功能是必不可少的早期发展和维持细胞活力，而其其他功能，当失去时，导致恶性表型。为了验证这一假设，我们的具体目标是：扩展我们对CTCF在肿瘤发生（目标1）和早期胚胎发育（目标2）中的作用的研究。我们将确定什么样的遗传和表观遗传机制参与CTCF单倍不足。最后（目的3），我们将使用Cre-loxP系统来产生条件性CTCF突变等位基因，其与各种组织特异性Cre转基因小鼠组合将允许我们测试以下假设：CTCF功能的完全丧失导致正常和/或恶性细胞中的细胞死亡，而体组织中一个CTCF等位基因的丧失易患肿瘤发展。我们还将产生携带我们在人类肿瘤中观察到的CTCF锌指点突变的敲入小鼠，以确定此类突变等位基因在肿瘤易感性中的功能意义。这一建议的意义在于，确定CTCF单倍不足的机制将为与CTCF图谱中16 q22.1处的洛缺失相关的临床人类癌症表型的广谱性提供新的见解。与健康相关的是，所确定的机制可以作为癌症预防和治疗干预的目标。